A. K. Dadayan

Solid State Isotope Exchange with Spillover Hydrogen in Organic Compounds

The term spillover, as used in heterogeneous catalysis, refers to the transport of active particles which are either absorbed or formed in one phase and transported to another phase where, under these reaction conditions, such particles are not absorbed or formed. An example is that of hydrogen, which after dissociative adsorption on platinum particles can migrate onto a nonorganic support such as aluminum oxide, barium sulfate, or others. Such active hydrogen atoms were given the name spillover hydrogen (SH).1 The first direct evidence of spillover was obtained during the reduction of wolfram trioxide to tungsten bronze at room temperature:2 the reaction proceeds in a mechanical mixture of 0.5% Pt/ Al2O3 + WO3. It was assumed that the hydrogen dissociated on the platinum and migrated through the alumina onto WO3 in the form of atoms or H+ ions. The solid state hydrogenation of asymmetric crystals of 2-isopropyl-5-methylphenol (also known as thymol) occurs with the participation of SH and leads to the formation of a series of asymmetric menthols and menthones.3 Despite the fact that processes involving SH have been known for over 40 years, the nature of this phenomenon has not been fully investigated or clarified. According to various hypotheses, hydrogen can migrate in the form of a solvated proton,4 as a proton-electron pair,5 or as atomic hydrogen.6 The main difficulty in explaining the SH phenomenon is that the SH concentration is too small to detect using direct instrumental analysis such as modern spectroscopic methods. Because the importance of SH in heterogeneous catalysis cannot be underestimated, the debate regarding the nature of the activated hydrogen particles and their diffusion is still ongoing.1

Evenly tritium labeled peptides in study of peptide in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a μg scale of biological samples both in vitro and in vivo.

Solid phase isotope exchange of deuterium and tritium for hydrogen in human recombinant insulin

Reaction of a high-temperature solid-phase catalytic isotope exchange in peptides and proteins under the action of the catalytically activated spillover hydrogen was studied. The reaction of human recombinant insulin with deuterium and tritium at 120–140°C resulted in an incorporation of 2–6 isotope hydrogen atoms per one insulin molecule. The distribution of the isotopic label by amino acid residues of the tritium-labeled insulin was determined by the oxidation of the protein S-S-bonds by performic acid, separation of polypeptide chains, their subsequent acidic hydrolysis, amino acid analysis, and liquid scintillation counts of tritium in the amino acids. The isotopic label was shown to be incorporated in all the amino acid residues of the protein, but the higher inclusion was observed for the FVNQHLCGSHLVE peptide fragment (B1–13) of the insulin B-chain, and the His5 and His10 residues of this fragment contained approximately 45% of the whole isotopic label of the protein. Reduction of the S-S-bonds by 2-mercaptoethanol, enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius, and HPLC fractionation of the obtained peptides were also used for the analysis of the distribution of the isotopic label in the peptide fragments of the labeled insulin. Peptide fragments which were formed after the hydrolysis of the Glu-Xaa bond of the B-chain were identified by mass spectrometry. The mass spectrometric analysis of the isotopomeric composition of the deuterium-labeled insulin demonstrated that all the protein molecules participated equally in the reaction of the solid-phase hydrogen isotope exchange. The tritium-labeled insulin preserved the complete physiological activity.

Ligands of glutamate and dopamine receptors evenly labeled with hydrogen isotopes

AbstactA reaction of high-temperature solid-phase catalytic isotope exchange (HSCIE) was studied for the preparation of tritium- and deuterium-labeled ligands of glutamate and dopamine receptors. Tritium-labeled (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclopenten-5,1-imine ([G-3H]MK-801) and R(+)-7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([G-3H]-7-OH-DPAT) were obtained with a specific activity of 210 and 120 Ci/mol, respectively. The isotopomeric distribution of deuterium-labeled ligands was studied using time-of-flight mass-spectrometer MX 5310 (ESI-o-TOF) with electrospray and orthogonal ion injection. Mean deuterium incorporation per ligand molecule was 11.09 and 3.21 atoms for [G-3H]MK-801 and [G-3H]-7-OH-DPAT, respectively. The isotope label was shown to be distributed all over the ligand molecule. The radioreceptor binding of tritium-labeled ligands [G-3H]MK-801 and [G-3H]-7-OH-DPAT was analyzed using the brain structure of Vistar rats. It was demonstrated that [G-3H]MK-801 specifically binds to hippocampus membranes with Kd 8.3 ± 1.4 nM, Bmax being 3345 ± 300 fmol/mg protein. The [G-3H]-7-OH-DPAT ligand specifically binds to rat striatum membranes with Kd 10.01 ± 0.91 nM and Bmax 125 ± 4.5 fmol/mg protein. It was concluded that the HSCIE reaction can be used for the preparation of highly tritium-labeled (+)-MK-801 and 7-OH-DPAT with retention of their physiological activities.

A comparative analysis of the distribution of glyprolines after their administration by different ways

The distribution of the glyprolines, Pro-Gly-Pro and Thr-Lys-Pro-Arg-Pro-Gly-Pro (Selanc), was analyzed and compared in tissues of rat organs after different ways of their administration using the peptides uniformly labeled with tritium. Comparative data on changes of concentrations of the peptides in the rat organs after their intraperitoneal, intranasal, intragastric, and intravenous administration are given. The intranasal administration of both peptides was shown to be optimal for delivery of glyprolines molecules in the CNS. A high affinity of the studied glyprolines for gastric tissues was found for all the ways of their administration. We suggest that high efficacy of action of glyprolines on homeostasis of the gastric mucosa was partially provided by accumulation of these peptides (to high concentrations) in gastric tissues.

Use of deuterium labeling by high‐temperature solid‐state hydrogen‐exchange reaction for mass spectrometric analysis of bradykinin biotransformation

RATIONALE Studies of molecular biodegradation by mass spectrometry often require synthetic compounds labeled with stable isotopes as internal standards. However, labeling is very expensive especially when a large number of compounds are needed for analysis of biotransformation. Here we describe an approach for qualitative and quantitative analysis using bradykinin (BK) and its in vitro degradation metabolites as an example. Its novelty lies in the use of deuterated peptides which are obtained by a high-temperature solid-state exchange (HSCIE) reaction. METHODS Deuterated and native BK were analyzed by positive electrospray ionization high-resolution mass spectrometry (ESI-HRMS) using an Orbitrap Fusion mass spectrometer. High-energy collision-induced dissociation (HCD) experiments were performed on [M+H](+) and [M+2H](2+) ions in targeted-MS(2) mode with adjusted normalized HCD value. RESULTS After the HSCIE reaction, each amino acid residue of the deuterated peptide contained deuterium atoms and the average degree of substitution was 5.5 atoms per the peptide molecule. The deuterated peptide demonstrated the same chromatographic mobility as the unlabeled counterpart, and lack of racemization during substitution with deuterium. Deuterium-labeled and unlabeled BKs were incubated with human plasma and their corresponding fragments BK(1-5) and BK(1-7), well known as the major metabolites, were detected. CONCLUSIONS Quantitative assays demonstrated applicability of the heavy peptide for both sequencing and quantification of generated fragments. Applicability of the HSCIE deuterated peptide for analysis of routes of its degradation has been shown in in vitro experiments. Copyright

Solid phase reaction of hemoglobin with spillover hydrogen

The reaction of high-temperature solid-state catalytic isotope exchange (HSCIE) between bovine hemoglobin and spillover hydrogen (SH) was studied. It was shown that, in the field of subunit contact, there is a significant decrease in ability for hydrogen exchange by SH. A comparison of the distribution of the isotope label in the hemoglobin α-subunit was carried out for the HSCIE reaction with the hemoglobin complex and with the free α-subunit. To this end, enzymatic hydrolysis of protein under the action of trypsin was carried out. The separation of tritium-labeled tryptic peptides was achieved by HPLC. Changes in availability of polypeptide chain fragments caused by complex formation were calculated using a molecular model. The formation of the protein complex was shown to lead to a decrease in the ability of fragments of α-subunits MFLSFPTTK (A32−40) and VDPVNFK (A93−99) for hydrogen replacement by tritium by almost an order of magnitude; hence, their availability to water (1.4 Å) twice decreased on the average. The decrease in ability to an exchange of hydrogen by spillover tritium on the formation of hemoglobin complex was shown to be connected with a reduction in availability of polypeptide chain fragments participating in spatial interactions of subunits with each other. Thus, the HSCIE reaction can be used not only for the preparative obtaining of tritium-labeled compounds, but also for determining the contact area in the formation of protein complexes.

Anxiolytic activity of the neuroprotective peptide HLDF-6 and its effects on brain neurotransmitter systems in BALB/c and C57BL/6 mice

This study is focused on a new amide derivative of the peptide HLDF-6 (Thr-Gly-Glu-Asn-His-Arg). This hexapeptide is a fragment of Human Leukaemia Differentiation Factor (HLDF). It displays a broad range of nootropic and neuroprotective activities. We showed, for the first time, that the peptide HLDF-6-amide has high anxiolytic activity. We used ‘open field’ and ‘elevated plus maze’ tests to demonstrate anxiolytic effects of HLDF-6-amide (0.1 and 0.3 mg/kg intranasally), which were comparable to those of the reference drug diazepam (0.5 mg/kg). Five daily equipotent doses of HLDF-6-amide selectively mitigated anxiety and increased the density of NMDA receptors in the hippocampus of stress-susceptible BALB/c mice, and had no effect on stress-resilient C57BL/6 mice. The subchronic administration of HLDF-6-amide showed no effect on the density of GABAA and nicotine receptors but was accompanied by a nonselective decrease of the 5-HT2A serotonin receptor density in frontal cortex of both strains. The mechanism of the specific anxiolytic activity of HLDF-6-amide may include its action on the NMDA-glutamatergic receptor system of the hippocampus and on serotonin 5-HT2A-receptors in the prefrontal cortex. The psychotropic activity of HLDF-6-amide is promising for its introduction to medical practice as a highly effective anxiolytic medicine for mental and neurological diseases.

[The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives].

The novel method for the peptide pharmacokinetics in tissues of laboratory animals was elaborated by the example of the HLDF-6 peptide amide. This method practically completely prevented the proteolytic degradation of peptides in the course of the analysis. The HLDF-6 hexapeptide (TGENHR) is a fragment corresponding to the 41–46 sequence of the human leukocyte differentiation factor (HLDF). It exhibits a wide spectrum of nootropic and neuroprotective activity. Therapeutic agents for prevention and therapy of cerebrovascular and neurodegenerative diseases have been created on the basis of the HLDF-6 amide (TGENHR-NH2). Pharmacokinetics and the molecular mechanism of action of the HLDF-6 peptide amide were studied using its tritium-labeled and deuterium-labeled derivatives. The labeled peptides were prepared with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). The tritiumlabeled [3H]TGENHR-NH2 peptide was obtained with a molar radioactivity of 230 Ci/mmol. The deuteriumlabeled [2H]TGENHR-NH2 peptide was prepared with an average deuterium incorporation of 10.5 atoms per the one peptide molecule. The NMR spectroscopy confirmed a uniform distribution of the isotope label throughout the whole peptide molecule. This uniformity allowed a qualitative analysis of both the peptide itself and all the possible fragments of its biodegradation in the organism’s tissues. The main TGENHR-NH2 metabolites which were formed during its proteolytic cleavage in the blood plasma were quantitatively analyzed and pharmacokinetics of the peptide amide was investigated with the use of its tritium-labeled derivative after intravenous and intranasal administration in mice, rats, and rabbits. Values of the basic pharmacokinetic parameters were calculated, the hypothesis of pharmacokinetic linearity was checked, and metabolism of the peptide was studied on the basis of the obtained pharmacokinetic profiles of TGENHR-NH2. The TGENHR-NH2 peptide was shown to have extremely high bioavailability with its intranasal administration (34% for rats). The peptide was quickly disappeared from blood due to its active proteolytic degradation in organism’s tissues. The TGENHR-NH2 peptide was shown to be highly stable towards the proteolytic hydrolysis during its incubation with the blood plasma, and a quantitative analysis of the formed metabolites was performed.